Methods for producing interfering rna molecules in mammalian cells and therapeutic uses for such molecules

ABSTRACT

Methods for producing interfering RNA molecules in mammalian cells are provided. Therapeutic uses for the expressed molecules, including inhibiting expression of HIV, are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 10/365,463 filed 13 Feb. 2003. U.S. patent application Ser. No.10/365,643 is related to and claims priority under 35 U.S.C. §119(e) toU.S. provisional patent application Ser. No. 60/356,127, filed 14 Feb.2002. Each of these applications is incorporated herein by reference.

GOVERNMENT RIGHTS STATEMENT

This invention was made with government support under Grant No. A1 29329awarded by the National Institutes of Health. The United Statesgovernment may have certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to RNA interference. More particularly,the present invention relates to methods for producing interfering RNAmolecules in mammalian cells, and genetic and therapeutic uses for suchexpressed molecules.

BACKGROUND OF THE INVENTION

RNA interference is the process of sequence-specific,post-transcriptional gene silencing in animals and plants initiated bydouble stranded (ds) RNA that is homologous to the silenced gene(Hammond, S. M. et al., 2000; Fire, A., 1999; Sharp, P. A., 2001). Inparticular, synthetic and endogenous siRNAs are known to direct targetedmRNA degradation (Hammond, S. M. et al., 2000; Elbashir, S. M. et al.,2001; Caplen, N. J. et al., 2001; Clemens, J. C. et al., 2000; Lipardi,C. et al., 2001; Elbashir, S. M. et al., 2001; Ui-Tei, K. et al., 2000).

This powerful genetic technology has usually involved injection ortransfection of ds RNA in model organisms. RNA interference also is apotent inhibitor of targeted gene expression in a variety of organisms(Wianny, F. et al., 2000; Kennerdell, J. R. et al., 1998; Fire, A. etal., 1998; Oelgeschlager, M. et al., 2000; Svoboda, P. et al., 2000).Recent studies by several groups (Lipardi, C. et al., 2001; Sijen, T. etal., 2001) suggest that ds small interfering RNAs (siRNAs) are part of ariboprotein complex that includes an RNAse III-related nuclease (Dicer)(Bernstein, E. et al., 2001), a helicase family (Dalmay, T. et al.,2001; Cogoni, C. et al., 1999), and possibly a kinase (Nykanen, A. etal., 2001) and an RdRP (Lipardi, C. et al., 2001; Smardon, A. et al.,2000). The mechanism proposed by Lipardi et al. (Lipardi, C. et al.,2001) is that one of the siRNA oligomers (antisense to the target RNA)primes an RdRP, generating longer dsRNAs, which are then cleaved by theRNAse III activity into additional siRNA duplexes, thereby amplifyingthe siRNAs from the target template.

dsRNA ≧30 by can trigger in mammalian cells interferon responses thatare intrinsically sequence-nonspecific (Elbashir, S. M. et al., 2001).However, duplexes of 21-nucleotide (nt) siRNAs with short 3′ overhangscan mediate RNA interference in a sequence-specific manner in culturedmammalian cells (Elbashir, S. M. et al., 2001). Two groups havedemonstrated that 19 to 21 base duplexes with 3′UU or TT overhangs caneffectively elicit an siRNA response in mammalian cells (Elbashir, S. M.et al., 2001; Caplen, N. J. et al., 2001). However, one limitation tothe use of siRNA as a therapeutic reagent in vertebrate cells is thatshort, highly defined RNAs need to be delivered to target cells, whichthus far has been accomplished only by using synthetic, duplexed RNAsdelivered exogenously to cells (Elbashir, S. M. et al., 2001; Caplen, N.J. et al., 2001).

The present invention overcomes at least the above limitation.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods for producingdouble stranded, interfering RNA molecules in mammalian cells, andpreferably human cells, by introducing into the cells DNA sequencesencoding the interfering RNA molecules.

In another aspect, the method comprises a) inserting DNA sequencesencoding a sense strand and an antisense strand of an interfering RNAmolecule into a vector comprising a suitable promoter, preferably a RNApol III promoter, and b) introducing the vector into a mammalian cell sothat the RNA molecule can be expressed.

In a preferred embodiment, the present invention includes firstselecting a target sequence, which preferably is accessible to thepairing between the target sequence and interfering RNA required for theinterfering RNA to function properly. Methods for identifying targetsites may be carried out using synthetic DNA oligonucleotides in cellextracts and/or a site selection approach on native RNAs, as describedherein. Once an optimal target site has been identified, the appropriatesequences for making the sense and antisense strands of the interferingRNA molecule can be synthesized.

Possible target sites include those found on the transcription productsof cellular or infectious agent genes (viral, bacterial etc.).

In another preferred embodiment, the RNA molecule produced is a smallinterfering RNA (siRNA) molecule, while the DNA sequences encoding thesense and antisense strands of the siRNA are siDNA.

In another preferred embodiment, the RNA pol III promoter is a mammalianU6 promoter, and more preferably the human U6 RNA Pol III promoter.

In another aspect, the invention provides methods for inhibiting theexpression of target genes, comprising introducing one or more vectorsinto a mammalian cell, wherein the one or more vectors comprise asuitable promoter and DNA sequences encoding a sense strand and anantisense strand of an interfering RNA, preferably siRNA, molecule. Theinterfering RNA molecule, which preferably is specific for thetranscription product of the target gene, can be then expressed andinitiate RNA interference of protein expression of the target gene inthe mammalian cell, thereby inhibiting expression of the target gene.

In another aspect, the invention provides a method for testing theexpression and function of siRNA molecules, comprising co-introducinginto a mammalian cell i) one or more vectors comprising a suitable firstpromoter and DNA sequences encoding a sense strand and an antisensestrand of an siRNA molecule, and ii) a vector comprising a target geneand a suitable second promoter. The siRNA molecule can be then expressedand initiate RNA interference of expression of the target gene, therebypotentially inhibiting expression of the target gene. Thus, theendogenous expression and function of the siRNA molecule can be assayedbased on the presence, if any, of RNA interference and more particularlyby any inhibition of expression of the target gene.

The present invention thus provides many possible therapeuticapplications, based on the design of the siRNA molecules and theirspecificity for selected disease targets. For example, one applicationof the invention is the treatment of HIV, for which siRNA molecules maybe designed to inhibit the expression of selected HIV targets, thusinhibiting HIV expression.

In a preferred embodiment, the invention provides a method forinhibiting expression of an HIV target gene, comprising introducing oneor more vectors into a mammalian cell, preferably an HIV-infected humancell. The one or more vectors comprise a suitable promoter and DNAsequences encoding a sense strand and an antisense strand of an siRNAmolecule, which preferably is specific for the transcription product ofthe HIV target gene. More preferably, the siRNA molecule is specific fora selected target site on the transcription product of the selected HIVtarget gene. The siRNA molecule can be then expressed and initiate RNAinterference of expression of the target gene, thereby inhibitingexpression of the target gene.

In a more preferred embodiment, the HIV is HIV-1. In another preferredembodiment, multiple siRNA constructs targeted to different sites in theHIV genome may be expressed, thereby initiating RNA interference ofexpression of several different HIV target genes and thus possiblycircumventing genetic resistance of the virus.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows a schematic diagram of a target rev-EGFP construct.

FIGS. 1B (SEQ ID NO:9) and 1C (AS(I):SEQ ID NO:1; S(I):SEQ ID NO:2;AS(II):SEQ ID NO:3; S(II):SEQ ID NO:4) show schematic diagrams of a U6promoter construct and a U6 promoter driven siRNA construct.

FIG. 1D shows siRNAs (S(I) (SEQ ID NO:5), AS(I) (SEQ ID NO:6), S(II)(SEQ ID NO:7), AS(II) (SEQ ID NO:8) in accordance with an embodiment ofthe invention.

FIG. 2 shows gel photographs from accessibility assays for sites I andII in cell extracts prepared from rev-EGFP expressing cells.

FIG. 3 shows photographs obtained from fluorescent microscope imaging ofthe effect of siRNA on EGFP expression.

FIG. 4 is bar graph showing the extent of inhibition of EGFP expressionby siRNAs in accordance with an embodiment of the invention.

FIGS. 5A to 5G show autoradiographs of Northern gel analyses.

FIG. 6 is a graph showing inhibition of HIV-1 NL4-3 by siRNAs inaccordance with an embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Interfering RNA molecules, and more preferably siRNA molecules, producedand/or used in accordance with the invention include those types knownin the art. The interfering RNA, and preferably siRNA, molecules aredouble-stranded (ds) RNAs that preferably contain about 19 to 23 basepairs. The molecules also may contain 3′ overhangs, preferably 3′UU or3′TT overhangs.

The term “introducing” encompasses a variety of methods of introducingDNA into a cell, either in vitro or in vivo, such methods includingtransformation, transduction, transfection, and infection. Vectors areuseful and preferred agents for introducing DNA encoding the interferingRNA molecules into cells. Possible vectors include plasmid vectors andviral vectors. Viral vectors include retroviral vectors, lentiviralvectors, or other vectors such as adenoviral vectors or adeno-associatedvectors.

In one embodiment, the DNA sequences are included in separate vectors,while in another embodiment, the DNA sequences are included in the samevector. If the DNA sequences are included in the same vector, the DNAsequences may also be inserted into the same transcriptional cassette.

Alternative delivery systems for introducing DNA into cells may also beused in the present invention, including, for example, liposomes, aswell as other delivery systems known in the art.

Suitable promoters include those promoters that promote expression ofthe interfering RNA molecules once operatively associated or linked withsequences encoding the RNA molecules. Such promoters include cellularpromoters and viral promoters, as known in the art. In one embodiment,the promoter is a RNA pol III promoter, which preferably is locatedimmediately upstream of the DNA sequences encoding the interfering RNAmolecule. Various viral promoters may be used, including, but notlimited to, the viral LTR, as well as adenovirus, SV40, and CMVpromoters, as known in the art.

In a preferred embodiment, the invention uses a mammalian U6 RNA Pol IIIpromoter, and more preferably the human U6snRNA Pol III promoter, whichhas been used previously for expression of short, defined ribozymetranscripts in human cells (Bertrand, E. et al., 1997; Good, P. D. etal., 1997). The U6 Pol III promoter and its simple termination sequence(four to six uridines) were found to express siRNAs in cells.Appropriately selected interfering RNA or siRNA encoding sequences canbe inserted into a transcriptional cassette, providing an optimal systemfor testing endogenous expression and function of the RNA molecules.

In a preferred embodiment, the mammalian cells are human cells. However,it is also understood that the invention may be carried out in othertarget cells, such as other types of vertebrate cells or eukaryoticcells.

In accordance with the invention, effective expression of siRNA duplexestargeted against the HIV-1 rev sequence was demonstrated. Using arev-EGFP (enhanced green fluorescent protein) fusion construct intransient co-transfection assays, ca 90% inhibition of expression wasobserved. The same siRNA expression constructs have been tested againstHIV in co-transfection assays resulting in a four-log reduction in HIVp24 antigen levels.

The above results were achieved using a human U6 snRNA Pol III promoterto express the appropriate 21 base oligomer RNAs in human cells. Thepromoter design is such that the first base of the transcript is thefirst base of the siRNA, and the transcript terminates within a run of 6U's encoded in the gene. The U6+1 promoter initiates transcription witha tri-phosphate, and the transcript is not capped unless the first 27bases of the U6 RNA are included in the transcript (Bertrand, E. et al.,1997; Good, P. D. et al., 1997). Thus, it was believed that siRNAs couldbe made whose structure would closely mimic certain predefinedrequirements (Elbashir, S. M. et al., 2001; Caplen, N. J. et al., 2001).

As stated above, expression cassettes are designed such that sequencesencoding sense and antisense strands of the siRNA can be in either thesame or separate vectors. Although the vector containing both sense andantisense strands was predicted to be superior to co-transfecting thetwo separately, this was not the case. It is likely that theco-transfection juxtaposes the two sequences so that transcripts haveample opportunity to form dsRNAs. An interesting feature of theexpression system is that in cells expressing both sense and antisenseRNA oligomers, an unexpected aberrantly sized product accumulates inlarge amounts (FIGS. 5A-D). Experiments with RNAse pretreatment of theRNAs prior to electrophoresis and blotting suggest that this largertranscript is double stranded. The ds RNA may be in the form of a simpleduplex, or could be covalently joined. Covalently linked siRNAs havebeen shown to be effective when expressed in cells, a result somewhatcontradictory to the results when using ex vivo delivered siRNAs(Elbashir, S. M. et al., 2001; Caplen, N. J. et al., 2001).

In order to ascertain whether or not there are differences in targetsite accessibilities for siRNA pairing as observed for antisense oligosand ribozymes (Scherr, M. et al., 1998; Scherr, M. et al., 2001;co-pending U.S. application Ser. No. 09/536,393, filed Mar. 28, 2000),two target sites for the siRNAs were tested. One site was chosen by anoligonucleotide library scanning mechanism designed to identify sitesaccessible to antisense pairing on native RNAs in cell extracts (Scherr,M. et al., 2001), whereas the other site was chosen at random in asegment of rev that overlaps with the HIV-1 tat sequence. Markeddifferences in accessibility to oligo pairing to these two sitestranslated to marked differences in siRNA inhibitory activities in therev-EGFP fusion. Despite the differences in potency against the rev-EGFPtarget, both siRNAs were potent inhibitors in HIV-1 co-transfectionassays. The invention thus demonstrates functional intracellularexpression of siRNAs in mammalian cells, particularly human cells.

An interesting result was the relationship between antisense DNAoligomer site-accessibility and the efficacy of the siRNAs targetedagainst the rev-EGFP transcript. More specifically, there was observed arelative lack of inhibition of the rev-EGFP target mediated by site IsiRNAs. This result was not the result of poor expression of theseoligomers, since they appear to be expressed in equivalent amounts tosite II siRNAs (FIGS. 5A-D). These differing results could be due to theposition of the siRNA target site relative to the end of the targettranscript, which has been demonstrated to limit amplification of thesiRNAs in Drosophila (Elbashir, S. M. et al., 2001). However, this doesnot seem to be the case since site I is positioned 301 nts downstream ofthe pIND-rev-EGFP transcriptional start site, which is well beyond theminimal distance required to amplify the siRNAs. These results could bedue to different targets used in different experiments. The relativeaccessibility of the site I in the context of the rev-EGFP fusion mRNAmay be limiting as shown by oligo-RNAseH experiments (FIG. 2). In theHIV-1 transcripts, site I is present in both the tat and revtranscripts, as well as in the singly spliced and unspliced transcripts.Given the complexity of different transcripts harboring site I, it isnot possible to state which of these is sensitive to the site I siRNAs.

Target mRNA for testing siRNA. A prerequisite for development of siRNAapproaches to silence viral gene expression is to have an appropriatehuman cell assay system. In order to assay the siRNAs, rev was fused toEGFP (enhanced green fluorescent protein) to provide a reporter systemfor monitoring siRNA function (FIG. 1A). Temporal control of target mRNAexpression was obtained by inserting the rev-EGFP fusion gene in theEcdysone-inducible pIND vector system (Invitrogen) (FIG. 1A). It will bereadily apparent to persons skilled in the art that alternative vectorsystems and promoters may be used for expressing selected target genesor target fusion genes during co-transfection assays.

In order to use the inducible system, a 293/EcR cell line was used,which was engineered to respond to the insect hormone analoguePonasterone A. When the pIND-rev-EGFP vector was transfected into thesecells followed by addition of the inducer, EGFP fluorescence wasobserved as early as 3 hr after addition of ponasterone A and continuedfor more than 100 hr. In the absence of ponasterone A, EGFP fluorescencewas not observed.

In FIG. 1A, the relative locations of the two siRNA target sites in therev-EGFP target are indicated, as are the locations of these two targetsites in HIV transcripts from pNL4-3.

Target site accessibility in the rev-EGFP fusion transcript. It waspreviously demonstrated that synthetic DNA oligonucleotides in cell orovary extracts can be used to identify sites accessible to base pairingby both antisense DNA and ribozymes (Scherr, M. et al., 1998; Scherr, M.et al., 2001; Lee, N. S. et al., 2001). Semi-random 19-mer DNA oligomerlibraries (Scherr, M. et al., 2001) were used in cell extracts preparedfrom cells expressing the rev-EGFP mRNA transcripts. Using this approachto screen the entire rev sequence, only a single TdPCR product wasidentified (data not shown), which centered within the sequence5′GCCTGTGCCTCT TCAGCTACC 3′ (SEQ ID NO:10) (site II), located 213 ntsdownstream from the AUG codon of rev and 494 nts downstream of the siteof pIND transcription initiation. Since this sequence harbors a CUChammerhead ribozyme cleavage motif, a hammerhead ribozyme was alsosynthesized that cleaves after the CUC site. This enabled a comparisonbetween the inhibitory activity of the siRNA with a ribozyme expressedfrom the same promoter system. To determine whether or not there aredifferences in the siRNA mediated targeting of a given message, a second21 nt site with a 5′ G and 3′ C was selected, which has a total GCcontent similar to site II. The requirements for a 5′ G and 3′ C arebased on the first nucleotide of the pTZU6+1 transcript, which initiateswith a G (FIGS. 1C and 1D). The second target sequence,5′GCGGAGACAGCGACGAA GAGC3′ (SEQ ID NO:11) (site I), is also located inan exon common to tat and rev, 20 nts downstream of the revtranslational initiation codon, and 301 nts downstream of the pINDtranscriptional initiation signal.

In FIG. 1B, the schematic presentation of the upstream promoter andtranscript portion of the U6 expression cassette is shown with thesequences and depicted structure of the expected primary transcript. InFIG. 1C, the sequences of the 21 base sense and antisense inserts with astring of 6T's and XbaI are shown. The first G came from the mungbean-treated SalI of pTZU6+1 vector. The 6T's may be processed to the2T's (capital letters) by the Pol III RNA polymerase. In FIG. 1D, theputative siRNAs derived from co-expression of the sense and antisense 21mers [S/AS (I) or (II)], with 3′ UU overhangs are depicted.

To determine whether the two target sequences were equally accessible toantisense pairing, two 21-mer DNA oligonucleotides complementary to eachof the two siRNA target sites were synthesized and used as probes foraccessibility to base pairing with the rev-EGFP fusion transcripts incell extracts. It was demonstrated (FIG. 2) that site II is highlyaccessible to base pairing with its cognate oligo (89% reduction inRT-PCR product relative to the no oligo control). This was in contrastto the results obtained with the site I oligo, which reduced therev-EGFP transcript by 27% relative to the control. Since these twosites have marked differences in their accessibilities to antisensepairing (FIG. 2), they provided a good test for the role that targetaccessibility plays in siRNA-mediated targeting.

In FIG. 2, the ethidium bromide-stained bands represent RT-PCR productsfrom rev-EGFP (top, 673 nt) or beta Actin (bottom, 348 nt) mRNAs. Thelanes from left to right are: control, no added oligo, minus (−) or plus(+) RT; oligonucleotide probing for site I (−) or (+) RT;oligonucleotide probing of site II (−) or (+) RT. The reduction intarget mRNA is elicited by endogenous RNAse H activity as describedpreviously (Scherr, M. et al., 1998).

Genes encoding siRNAs targeted to site I or II were inserted behind thePol III U6 SnRNA promoter of pTZU6+1 (FIGS. 1B and 1C). Thetranscriptional cassettes were constructed such that they are either inthe same or different vectors. The constructs in separate vectorsprovided a set of sense and antisense controls.

Reduction of target gene expression. The siRNA sequences, along withsense, antisense, or ribozyme controls were cotransfected with thetarget rev-EGFP expressing plasmid into 293/EcR cells. Sixteen to twentyhours later, the inducer Ponasterone A was added to the cell culturesresulting in induction of the rev-EGFP fusion product. The cells wereincubated an additional 48 hours prior to fluorescent microscopicanalyses and fluorescence activated cell sorting (FACS). Combined senseand antisense RNA oligomers targeted to site II in the rev sequencereduced the EGFP signal by ca 90% relative to the controls, whereas thecombined sense and antisense RNA oligomers targeted against site I gaveonly a modest reduction in fluorescence (FIGS. 3 and 4). The inhibitionmediated by the site II siRNAs was similar regardless of whether bothsense and antisense RNA oligomers were expressed from the same plasmidor different plasmid backbones (see FIGS. 1B and 1C). The controlconstructs, which included sense alone, antisense alone or a ribozymetargeted to site II, each expressed from the U6 promoter, gave nosignificant reduction of EGFP expression relative to the vector backbonecontrol (FIGS. 3 and 4).

In FIG. 3, 293/EcR cells were co-transfected with pIND-rev-EGFP andvarious siRNA constructs as indicated. Cells were examinedmicroscopically for EGFP expression following Ponasterone A addition asdescribed herein. Panel E shows fluorescent cells after transfectionwith control which is an irrelevant sense/antisense construct to therev. Other types of controls [S(II), AS(II), vector] were similar (A, B,D). Panel C shows ˜90% reduction in fluorescent cells when 293/EcR cellswere transfected with S/AS(II). Panels F-J are DAPI-stained imagesshowing that the same number of cells are present in each field.Specific silencing of target genes was confirmed in at least threeindependent experiments.

In FIG. 4, 293/EcR cells were co-transfected with pIND-rev-EGFP andsiRNA constructs as described herein. Cells were analyzed for EGFPexpression by FACS and the level of fluorescence relative to cellstransfected with pIND-rev-EGFP alone was quantitated. Data are theaverage ±SD of 3 separate experiments. Only the siRNA constructcontaining both sense and antisense sequences directed at accessiblesite II [S/AS(II) or S+AS(II)] showed approximately 90% reductionrelative to the controls or vector only. The various combinations of U6driven siRNA constructs co-transfected with pIND-rev-EGFP are indicated.S/AS indicates the vector with both sense and antisense siRNA sequencewhile S+AS indicates the siRNA sequences in separate vectors. Rbzindicates the hammerhead ribozyme against site II. Specific silencing oftarget genes was confirmed in at least three independent experiments.

Expression of siRNAs and targets in 293 cells. Northern gel analyseswere carried out to examine the expression patterns and sizes of thesiRNAs transcribed from the U6 RNA Pol III promoter system in 293 cells.These Northern gel analyses demonstrated strong expression of sense andantisense RNAs as monitored by hybridization to the appropriate probes(FIG. 5).

In FIG. 5, RNA samples were prepared from 293/EcR cells transientlyco-transfected with pIND-rev-GFP and various siRNA constructs asindicated and subjected to Ponasterone A induction as described above.The total RNA was resolved on a 10% acrylamide/8M urea gel for siRNAs, a1% agarose/formamide gel for the target, or a 10% acrylamide/7M urea gelfor RNAse A/T1 treatment. In FIGS. 5A to 5D, hybridization was performedusing ³²P labeled DNA probes for sense or antisense transcripts for siteI and II siRNAs. The hybridizing products were ˜23- and ˜46-nts inlength. FIG. 5E shows the results of hybridization of the site IIdirected ribozyme (II) transcripts. RNAs prepared from cells expressingthe ribozyme for site II detected a transcript of the size expected forthe ribozyme transcript (˜75 nt). Since the probe used to detect theribozyme is also complementary to the antisense siRNA for site II, italso hybridized to the antisense RNAs targeted to site II.

The control RNAs (sense alone, antisense alone or ribozyme) were alldetected at the expected sizes (FIGS. 5A-E). RNAs prepared from cellssimultaneously expressing sense and antisense constructs generatedhybridized products of the sizes expected for the individual short RNAs(˜23 nts). In addition to the monomer sized RNAs, a strong hybridizationproduct approximately twice the size of the short RNA oligomers (˜46nts) was clearly visible (FIGS. 5A-E). This product was only detected inRNAs prepared from cells expressing both sense and antisense, andhybridized with both sense and antisense probes (FIGS. 5A-E). Since thegel system used to resolve the transcripts was a denaturing gel, itseemed unlikely that the aberrantly sized product could be dsRNA.Nevertheless, to test this possibility, the RNA samples were treatedwith a mixture of RNAse A and T1 prior to denaturing gel electrophoresisand blotting. Both of these RNAses preferentially cleave single strandedRNAs. Thus, if the aberrant product is double stranded, it should befully resistant to nuclease destruction. Two types of analyses wereperformed. The first involved simply treating the RNA samples with theRNAse mixture, whereas the second involved heating the samples to 95° C.prior to RNAse treatment (FIG. 5G).

In FIG. 5G, the RNAs from a combined transfection using S/AS(I+II) weretreated with a mixture of RNAse A and T1. Samples were either heated (+)or not heated (−) at 90° C. prior to RNAse treatment. The hybridizingproduct in the lane treated with RNAses, but without heat may be ˜21 ntsin length. This product was observed with probes for either sense orantisense siRNAs targeted to site II.

Fully duplexed RNAs should be resistant to cleavage, whereas heattreatment followed by quick cooling of the RNAs would separate thestrands and make them susceptible to RNAse cleavage. The resultsobtained (FIG. 5G) were consistent with the aberrantly sized productbeing double stranded. The non-heated sample treated with the RNAse mixgenerated a product migrating faster than the other RNAs. The fastermigrating RNA species hybridized to both the antisense and sense probes.In contrast, the sample that was heated prior to RNAse treatment gave nodetectable hybridizing bands. The faster migration of this product couldbe due to RNAse trimming of non-base paired ends of the duplex. Theseproducts could also derive from RNAse cleavage of single stranded loops,which would unlink the two RNAs, allowing for faster gel mobility.Importantly, the large, aberrantly sized transcripts are only generatedin cells expressing both sense and antisense transcripts, and thereforemust be dependent upon formation of ds RNAs (Bernstein, E. et al., 2001;Clemens, J. C. et al., 2000). In addition to the aberrantly sizedproduct, another band migrating between the aberrantly sized product andthe 106 nt U6 snRNA was detected only with a probe that is complementaryto the antisense siRNA for site II. This product was ˜65 nts in lengthand could be a product of the first dicer cleavage reaction on an RNAdependent RNA polymerase (RdRP) extended product of the siRNA antisense(Lipardi, C. et al., 2001; Sijen, T. et al., 2001). Finally, U6+1expression of the ribozyme targeted to site II (FIG. 5E) did not resultin inhibition of rev-EGFP expression (FIG. 4).

To determine whether siRNA complexes directed degradation of therev-EGFP mRNA, Northern hybridization analyses were carried out to probefor the rev-EGFP transcripts (FIG. 5F). FIG. 5F shows the results ofhybridization of the rev-EGFP fusion transcripts. Human GAPDH mRNA andU6snRNA were probed as internal controls for each experiment. These datademonstrated selective destruction of the fusion transcript only incells expressing the combination of site II sense and antisense siRNAs.The site I siRNAs, although abundantly expressed (FIGS. 5A and 5B)resulted in marginal inhibition of rev-EGFP expression (FIG. 4), withlittle degradation of the transcript (FIG. 5F). The combination of sitesI and II siRNAs resulted in less inhibition than the site II siRNAsalone. This is believed to result from a dosage effect in that theconcentration of each of the plasmids encoding these siRNAs was one halfthat used for the single site cotransfections. Most interestingly, thesite I siRNAs were potent inhibitors of HIV replication.

Inhibition of HIV by expressed siRNAs. In another embodiment of theinvention, an siRNA expression system was constructed for inhibitingHIV-1 infection. In order to test the potential inhibitory activity ofthe various constructs described above, each of the siRNA vectors (siteI and II) as well as the control constructs were co-transfected withHIV-1 pNL4-3 proviral DNA into 293 cells. At the intervals indicated inFIG. 6, supernatant samples were withdrawn from the cell cultures andHIV-1 p24 viral antigen levels were measured. In FIG. 6, pNL4-3 proviralDNA was cotransfected with the various U6+1 driven siRNA constructs at1:5 ratio of proviral DNA to U6 construct DNAs. Twenty-four hours posttransfection, and at the indicated times, supernatant aliquots werewithdrawn for HIV-1 p24 antigen assays. The various siRNA constructsused are indicated in FIG. 6.

The site II siRNAs were found to strongly inhibit HIV-1 replication inthis assay. Somewhat unexpectedly, the site I siRNAs also potentlyinhibited HIV-1 replication (as measured by p24 antigen production). Thecombination of both site I and II siRNA constructs was the most potent,providing ca four logs of inhibition relative to the control constructs.Such potent inhibition of HIV-1 has not been previously observed withother RNA-based anti-HIV-1 agents using a co-transfection assay.Possible explanations for the differences in inhibitory activity by thesite I siRNAs against the rev-EGFP fusion versus HIV-1 itself areaddressed herein. The observation that two different HIV targets areboth substrates for siRNA is highly encouraging for strategies requiringmultiple targeting to circumvent genetic resistance of the virus.

The present invention is further illustrated by the following exampleswhich are not intended to be limiting.

Example 1 Accessibility Assay

To evaluate the accessibility of target sequences for antisense basepairing, endogenous RNase H activity present in the cell extractsprepared from the stable 293 cells containing the rev-GFP was utilized.Two DNA oligonucleotides complementary to each of the two target siteswere synthesized and used as probes for accessibility in cell extractsaccording to the protocol of Scherr, M. et al., 1998, as describedherein, with some minor modifications.

Stable 293 cells containing CMV-revGFP gene were grown to 50 to 90%confluency in 100 mm dishes. Cells were harvested using a cell scraperand rinsed two times in phosphate buffered saline (PBS) twice. The cellswere resuspended in the same volume of hypotonic swelling buffer (7 mMTris-HCl, pH 7.5, 7 mM KCl, 1 mM MgCl₂, 1 mM β-mercaptoethanol) and1/10th of the final volume of neutralizing buffer (21 mM Tris-HCl, pH7.5, 116 mM KCl, 3.6 mM MgCl₂, 6 mM β-mercaptoethanol) on ice. Theresuspended solutions were sonicated 15 seconds three times in an icewater bath. The homogenate was centrifuged at 20,000×g for 10 minutes at4° C. The supernatants were used immediately, or stored as aliquots inthe same volume of glycerol storage buffer (15 mM Tris-HCl, pH7.5, 60 mMKCl, 2.5 mM MgCl₂, 45% glycerol, 5 mM β-mercaptoethanol) at −80° C.These frozen aliquots were always used within 3 months post freezing.

The cell extracts were incubated with 4 μM of the respective 21 byantisense oligodeoxyribonucleotides (site I or II) for 15 minutes at 37°C. in a total volume of 30 μl cleavage buffer (100 mM Tris-HCl, pH7.5,100 mM MgCl₂, and 10 mM DDT). After phenol extraction and ethanolprecipitation, the precipitates were digested with 20 U of DNase I for45 minutes at 37° C., followed by phenol extraction and ethanolprecipitation. The precipitates were resuspended in DEPC water andmonitored for OD₂₆₀ absorption.

The reverse transcriptase (RT) reaction was carried out using 300 ng to1 μg total RNA prepared from the above 30 μl extract sample with 5 U ofMoloney murine leukemia virus reverse transcriptase (Mo-MLV Rtase, LifeTechnologies, Inc. NY) according to the manufacturer's instructions.

First-strand priming was performed with 20 pmol of 3′ primer of an oligocomplementary to the rev sequence or 50 ng of random hexamer primers.β-actin was used as an internal control. PCR reactions for each set ofprimers were performed separately in a total volume of 50 μl containing10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.1 mM of each dNTP,0.4 μM of each primer, 1.5U of taq DNA polymerase and 2 μl of RTreaction sample. The PCR was carried out at 94° C. for 30 seconds, 68°C. for 30 seconds, and at 72° C. for 30 seconds for a total of 24 to 36cycles after cycle studying of control.

Reaction samples were separated on a 1.2% agarose gel and visualized byethidium bromide staining, then quantified using AlphaImager™quantitation software (Alpha Innotech Corp.).

Example 2 Constructs

A SacII (filled in)-EcoRI fragment containing the rev-GFP fusion gene ofCMV-rev-GFP was inserted into HindIII (filled in)-EcoRI sites of thepIND vector (Invitrogen), yielding the pIND-rev-GFP construct of (FIG.1A).

To construct the siRNA expression vectors, two cassettes were preparedusing the pTZ U6+1 vector (FIG. 1B). (Bertrand, E. et al., 1997; Good,P. D. et al., 1997). One cassette harbors the 21-nt sense sequences andthe other a 21 nt antisense sequence. These sequences were designed totarget either site I or site II (FIG. 1A). A string of 6T's was insertedat the 3′ terminus of each of the 21 mers followed by a XbaI restrictionsite. The 20-nt sense or antisense sequences (excluding the first G)with a string of 6T's and the XbaI restriction site were prepared fromsynthetic oligonucleotides (Lee, N. S. et al., 2001; Lee, N. S. et al.,1999). The first G of the inserts was provide by the SalI site of thevector which was rendered blunt-ended by mung bean nuclease. The insertswere digested by BsrBI for sense and AluI for antisense (site I), StuIfor sense and SnaBI for antisense (site II) for blunt end cloningimmediately downstream of the U6 promoter sequence. The 3′ ends of theinserts were digested with Xba I for insertion in the SalI (blunted)-XbaI digested pTZU6+1 vector to create the desired transcription units(FIG. 1C).

To create plasmids in which both sense and antisense sequences were inthe same vector, the pTZU6+1 sense sequence harboring vectors weredigested with BamHI (which was filled in using T4 DNA polymerase) andHindIII. The digested fragments containing the sense sequences weresubcloned into the SphI (filled in by T4 DNA polymerase)-HindIII sitesof the antisense AS(I) or AS(II) constructs, generating both sense andantisense transcription units [S/AS(I) or S/AS(II)] (FIG. 1C). The DNAsequences for each of the above constructs were confirmed prior to use.

Example 3 Cell Culture

293/EcR cells were grown at 37° C. in EMEM supplemented with 10% FBS, 2mM L-glutamine and 0.4 mg/ml of Zeocine. Twenty-four hour beforetransfection, cells were replated to 24- or 6-well plates at 50-70%confluency with fresh medium without Zeocine. Co-transfection of targetplasmids (pIND-rev-GFP) and siDNAs was carried out at 1:1 ratio withLipofectamine Plus™ reagent (Life Technologies, GibcoBRL) as describedby the manufacturer. 0.5 mg pIND-rev-GFP and 0.5 mg siDNAs and 0.1 mgpCMV-lacZ (for transfection efficiency), formulated into LipofectaminePlus, were applied per 6-well culture. Cells were incubated overnightand on the following day 5 mM Ponasterone A (Invitrogen) was added toinduce expression of pIND-rev-EGFP. Two days post induction thetransfected cells were harvested to measure EGFP fluorescence by FACSusing a modular flow cytometer (Cytomation, Fl). Transfectionefficiencies were normalized using a fluorescent b-galactosidase assay(Diagnostic Chemicals Ltd, CN). Fluorescent microscope imaging was alsoperformed to monitor EGFP expression. For the microscopic visualization,cells were grown on glass coverslips in 24-well plates. Co-transfectionswere carried out on glass coverslips using 0.25 mg each of rev-EGFP andsiRNA expression plasmid DNAs (total 0.5 mg). After 2 days, transfectedcells were fixed in 4% PFA for 15 minutes at room temperature andtreated with antifading reagent containing DAPI. Images were collectedusing an Olympus BX50 microscope and a DEI-750 video camera (Optronics)at 40× magnification with exposure time of ⅛ sec. Specific silencing oftarget genes was confirmed in at least three independent experiments.

Example 4 Northern Blotting

RNA samples were prepared from 293-EcR cells transiently co-transfectedwith pIND-rev-GFP and siRNAs and subjected to Ponasterone A induction asdescribed above. Total RNA isolation was preformed using the RNA STAT-60(TEL-TEST “B”) according to the manufacturer's instruction. The totalRNA was resolved on a 10% acrylamide/8M urea gel for siRNAs or a 1%agarose/formamide gel for the target, and transferred onto Hybond-N⁺membrane (Amersham Pharmacia Biotech). The hybridization and wash stepswere performed at 37° C. To detect the sense or antisense siRNAs,radiolabeled 21-mer DNA probes were used. Human U6 snRNA and GAPDH mRNAwere also probed for as internal standards. To detect the ribozymetargeted against site II of the rev mRNA, a 42-mer probe complementaryto the entire ribozyme core and flanking sequences was used (this probealso detects the antisense siRNA oligomer for site II). Forcharacterization of the aberrantly sized 46-nt RNAs, total RNAs fromS/AS (I+II) were treated with a mixture of RNAase A and T1 for 30minutes at 37° C. either with or without preheating at 90° C. for 5minutes.

For detection of the rev-EGFP mRNA, a 25-mer probe complementary to theEFP mRNA of the rev-GFP fusion protein was used.

Example 5 HIV-1 Antiviral Assay

For determination of anti-HIV-1 activity of the siRNAs, transient assayswere performed by cotransfection of siDNAs and infectious HIV-1 proviralDNA, pNL4-3 into 293 cells. Prior to transfection, the cells were grownfor 24 hours in six-well plates in 2 ml EMEM supplemented with 10% FBSand 2 mM L-glutamine, and transfected using Lipofectamine Plus™ reagent(Life Technologies, GibcoBRL) as described by the manufacturer. The DNAmixtures consisting of 0.8 μg siDNAs or controls, and 0.2 μg pNL4-3 wereformulated into cationic lipids and applied to the cells. After 1, 2, 3and 4 days, supernatants were collected and analyzed for HIV-1 p24antigen (Beckman Coulter Corp). The p24 values were calculated with theaid of the Dynatech MR5000 ELISA plate reader (Dynatech Lab Inc). Cellviability was also performed using a Trypan Blue dye exclusion count at4 days after transfection.

The above demonstrates the invention's utility for, among other things,designing and testing siRNA transcripts for a variety of genetic andtherapeutic applications. The invention also is believed to demonstratefor the first time the functional expression of siRNAs in mammaliancells.

The above results also demonstrate the utility of siRNAs as HIV-1inhibitory agents. By combining several siRNA constructs targeted todifferent sites in the HIV-1 genome, it should be possible to circumventgenetic resistance of the virus, thereby creating a potent gene therapyapproach for the treatment of HIV-1 infection.

The publications and other materials cited herein to illuminate thebackground of the invention and to provide additional details respectingthe practice of the invention are incorporated herein by reference tothe same extent as if they were individually indicated to beincorporated by reference.

While the invention has been disclosed by reference to the details ofpreferred embodiments of the invention, it is to be understood that thedisclosure is intended in an illustrative rather than a limiting sense,as it is contemplated that modifications will readily occur to thoseskilled in the art, within the spirit of the invention and the scope ofthe appended claims.

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1. An interfering RNA molecule comprising (i) a sequence complimentaryto a target sequence of an HIV-1 gene and (ii) a sequence complementaryto the sequence of (i), wherein sequence (i) has the sequenceGGUAGCUGAAGAGGCACAGGCUU (nucleotides 1-21 of SEQ ID NO:8) and whereinsequence (ii) is paired to sequence (i) to form a double stranded RNAmolecule.
 2. The interfering RNA molecule of claim 1, wherein sequence(i) has the sequence GGUAGCUGAAGAGGCACAGGCUU (SEQ ID NO:8)
 3. Theinterfering RNA molecule of claim 1, wherein sequence (ii) has thesequence GCCUGUGCCUCUUCAGCUACC (nucleotides 1-21 of SEQ ID NO:7).
 4. Theinterfering RNA molecule of claim 2, wherein sequence (ii) has thesequence GCCUGUGCCUCUUCAGCUACCUU (SEQ ID NO:7).
 5. A DNA sequenceencoding an antisense strand of an interfering RNA molecule, wherein theantisense strand targets an HIV-1 gene, can be expressed in a mammaliancell when introduced into the mammalian cell and when paired with acomplementary sense strand of the interfering RNA molecule can initiateRNA interference of expression of the HIV-1 target gene in the mammaliancell, thereby inhibiting expression of the HIV-1 target gene and whereinthe antisense strand of the interfering RNA molecule has the sequenceGGUAGCUGAAGAGGCACAGGC (nucleotides 1-21 of SEQ ID NO:8).
 6. The DNAsequence of claim 5, wherein sequence (i) has the sequenceGGUAGCUGAAGAGGCACAGGCUU (SEQ ID NO:8)
 7. The DNA sequence of claim 5,wherein the DNA sequence is operatively linked to a Pol III promoter. 8.The DNA sequence of claim 7, wherein the RNA pol III promoter is amammalian U6 RNA pol III promoter.
 9. A vector comprising the DNAsequence of claim
 5. 10. The vector of claim 9, wherein the vector is aviral vector.
 11. A vector comprising the DNA sequence of claim
 7. 12.The vector of claim 11, wherein the vector is a viral vector.
 13. Avector comprising the DNA sequence of claim
 8. 14. The vector of claim13, wherein the vector is a viral vector.
 15. An interfering RNAmolecule comprising (i) a sequence complimentary to a target sequence ofan HIV-1 gene and (ii) a sequence complementary to the sequence of (i),wherein sequence (i) has the sequence GCUCUUCGUCGCUGUCUCCGC (nucleotides1-21 of SEQ ID NO:6) and wherein sequence (ii) is paired to sequence (i)to form a double stranded RNA molecule.
 16. The interfering RNA moleculeof claim 15, wherein sequence (i) has the sequenceGCUCUUCGUCGCUGUCUCCGCUU (SEQ ID NO:6).
 17. The interfering RNA moleculeof claim 15, wherein sequence (ii) has the sequenceGCGGAGACAGCGACGAAGAGC (nucleotides 1-21 of SEQ ID NO:5).
 18. Theinterfering RNA molecule of claim 16, wherein sequence (ii) has thesequence GCGGAGACAGCGACGAAGAGCUU (SEQ ID NO:5).
 19. A DNA sequenceencoding an antisense strand of an interfering RNA molecule, wherein theantisense strand targets an HIV-1 gene, can be expressed in a mammaliancell when introduced into the mammalian cell and when paired with acomplementary sense strand of the interfering RNA molecule can initiateRNA interference of expression of the HIV-1 target gene in the mammaliancell, thereby inhibiting expression of the HIV-1 target gene and whereinthe antisense strand of the interfering RNA molecule has the sequenceGCUCUUCGUCGCUGUCUCCGC (nucleotides 1-21 of SEQ ID NO:6).
 20. The DNAsequence of claim 19, wherein sequence (i) has the sequenceGCUCUUCGUCGCUGUCUCCGCUU (SEQ ID NO:6).
 21. The DNA sequence of claim 19,wherein the DNA sequence is operatively linked to a Pol III promoter.22. The DNA sequence of claim 21, wherein the RNA pol III promoter is amammalian U6 RNA pol III promoter.
 23. A vector comprising the DNAsequence of claim
 19. 24. The vector of claim 23, wherein the vector isa viral vector.
 25. A vector comprising the DNA sequence of claim 21.26. The vector of claim 25, wherein the vector is a viral vector.
 27. Avector comprising the DNA sequence of claim
 22. 28. The vector of claim27, wherein the vector is a viral vector.